Biosensor, measuring instrument for biosensor, and method of quantifying substrate

ABSTRACT

A method of measuring a quantity of a substrate contained in sample liquid is provided. This method can reduce measurement errors caused by a biosensor. The biosensor includes at least a pair of electrodes on an insulating board and is inserted into a measuring device which includes a supporting section for supporting detachably the biosensor, plural connecting terminals to be coupled to the respective electrodes, and a driving power supply which applies a voltage to the respective electrodes via the connecting terminals. One of the electrodes of the biosensor is connected to the first and second connecting terminals of the measuring device only when the biosensor is inserted into the measuring device in a given direction, and has a structure such that the electrode becomes conductive between the first and second connecting terminals due to a voltage application by the driving power supply.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/182,236, filed Nov. 21, 2002, now U.S. Pat. No. 7,232,510, which is a35 U.S.C. §371 U.S. National Phase of PCT Application No.PCT/JP01/10525, filed Nov. 30, 2001, and claims priority to JapaneseApplication No. 2000-364225, filed Nov. 30, 2000, and JapaneseApplication No. 2001-357144, filed Nov. 22, 2001, the contents of all ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a biosensor for measuring the quantityof a substrate included in sample liquid and a measuring device for thebiosensor. Further, the present invention provides a novel measuringmethod which reduces measurement errors caused by a biosensor.

BACKGROUND ART

Biosensors measure the quantity of a substrate included in sampleliquid. The sensors utilize molecular recognition capability of biomaterial such as germ, enzyme, antibody, DNA, RNA and the like, and usesthe bio material as a molecular recognizing element. In other words,when the bio material recognizes an objective substrate, it reacts suchthat the germ breathes, emits light, consumes oxygen, or causes enzymereaction. The biosensors utilize those reactions and measure thequantity of the substrate included in the sample liquid. Among thebiosensors, enzyme sensors have been promoted to practical use. Forinstance, an enzyme sensor for glucose, lactic acid, cholesterol, andamino acid is used in medical measurement and food industry. The enzymesensor reduces an electron carrier with an electron produced by thereaction between the substrate and the enzyme included in the sampleliquid, i.e., specimen. A measuring device measures the reduced amountof the electron carrier electrochemically, so that quantative analysisof the specimen is carried out.

Various kinds of biosensors, such as the one discussed above, have beenproposed. A conventional biosensor, biosensor Z, will be describedhereinafter. FIG. 16( a) shows a perspective exploded view of biosensorZ. FIG. 16( b) shows a structure of an electrode formed at a tip ofbiosensor Z. A method of measuring a quantity of a substrate in a sampleliquid will be described with reference to FIG. 16( b).

First, biosensor Z is inserted into a measuring device. The measuringdevice applies a given voltage across counter electrode 1103 a andmeasuring electrode 1103 b. Then the sample liquid is supplied to inlet1106 b of a sample supplying path. The sample liquid is sucked into thesupplying path due to capillary phenomenon, and passes on counterelectrode 1103 a, which is nearer to inlet 1106 b, and arrives atmeasuring electrode 1103 b. Then reagent layer 1105 starts dissolving.At this time, the measuring device detects an electrical changeoccurring between counter electrode 1103 a and measuring electrode 1103b, and starts measuring the quantity. The quantity of the substrateincluded in the sample liquid is thus measured.

Specifically, oxidoreductase and an electron acceptor retained in thereagent layer dissolve into the sample liquid, and enzyme reactionprogresses between the substrate in the liquid. Then the electronacceptor is reduced. After the reaction finishes, the reduced electronacceptor is oxidized electrochemically. A concentration of the substratecan be measured using an oxidation current measured when the acceptor isoxidized.

However, the conventional biosensor Z has some problems to be solved. Inparticular, when the measuring device detects the electrical change inreagent layer 1105, various factors influence measurement accuracy andsensitivity of the measuring device.

First, an incorrect operation by a user influences them. For instance:(1) After the user supplies the sample liquid to the sample supplyingpath, the user adds another sample liquid before the measuring devicecompletes the measurement; (2) The user tries to measure the quantitywith a biosensor which have been already used; (3) The user supplies thesample liquid to a incorrect place; (4) The user inserts the biosensorinto the measuring device in a wrong direction; and (5) When supplyingthe sample liquid, the user fails to pinpoint an inlet of the samplesupplying path, has the sample liquid attach to a surrounding area, andthus has the sample liquid not run into the path. Thus some ways havebeen desired to avoid those incorrect operations which influence themeasurement accuracy. In particular, preventing aged users from theincorrect operations is required.

Second, characteristics of an object to be measured influence them. Forinstance, when a glucose concentration of human blood is measured with abiosensor, a viscosity of the blood may influence measurement accuracy.Hematocrit, which is generally known as an index of blood viscosity,indicates a volume percentage of erythrocyte included in the blood.Blood in a person who does not suffer from anemia includes 50-60 volume% of water and 40-50 volume % of erythrocyte. If suffering from renalanemia due to chronic renal failure, a person has blood have the volumepercentage of hematocrit decrease to less than 15%. Appropriatetreatment requires to restrain the influence to hematocrit in the bloodfor accurate measurement of glucose concentration in the blood of, e.g.,a diabetic.

Third, a temperature around the measuring device influences them.Measuring devices available in the market for biosensors have beendownsized so that users can carry it with them. Soon after moving intoindoors from the outside, a user may try to measure the quantity. Inthis case, the measurement may start before a temperature in themeasuring device becomes stable. A sharp change in temperatureinfluences the oxidation current corresponding to a substrateconcentration, and thus may lower the measurement accuracy. A bodytemperature of the user, upon being transmitted to the measuring devicevia, e.g., the user's hand, might influence the measurement accuracy.

The present invention thus aims to provide a biosensor being handledeasily and having excellent measurement accuracy, a method of measuringquantity using the biosensor, and a measuring device using thebiosensor.

SUMMARY OF THE INVENTION

For solving the above problems, a first aspect of the present inventionprovides a biosensor for measuring the quantity of a substrate includedin sample liquid. The biosensor is inserted to a measuring device whichincludes a supporting section for supporting detachably a biosensorwhich is formed of at least a pair of electrodes on an insulating board,plural connecting terminals electrically connected to the electrodesrespectively, and a driving power supply for applying a voltage to theelectrodes via the connecting terminals. One of the electrodes of thebiosensor is connected to first and second connecting terminals of themeasuring device only when the biosensor is inserted into the supportingsection of the measuring device in a given direction. Then, the one ofthe electrodes becomes conductive due to a voltage application by thedriving power supply. The electrodes have such a structure discussedabove.

A conductive layer may be formed on at least a part of the insulatingboard, and the conductive layer is divided by slits, thereby forming acounter electrode and a measuring electrode, and upon request, adetecting electrode may be also formed.

A second aspect of the present invention aims to provide a measuringdevice to be used with a biosensor, and to measures a quantity of asubstrate included in sample liquid. The measuring device includes asupporting section for supporting detachably the biosensor including atleast a pair of electrodes on an insulating board, plural connectingterminals electrically connected to the electrodes, respectively, and adriving power supply for applying a voltage to the electrodes via theconnecting terminals. The measuring device includes first and secondconnecting terminals can be connected to either one of electrodes of thebiosensor only when the biosensor is inserted into the supportingsection in a given direction. Thus conductivity can be detected betweenthe first and the second connecting terminals by applying a voltage fromthe driving power supply to the first and second terminals,respectively.

It is also possible that the measuring device may determine that thebiosensor is not inserted in the given direction if the conductivity isnot detected. It is also possible that the measuring device may includean output section which outputs the determination to outside when thedevice determines that the biosensor is not inserted in the givendirection.

A third aspect of the present invention provides a method of measuring aquantity of a substrate included in sample liquid with a biosensor. Thebiosensor includes an electrode section including: a counter electrode,a measuring electrode, and a detecting electrode on at least a part ofan insulating board; a sample supplying path for supplying the sampleliquid to the electrode section; and a reagent layer for reacting on thesample liquid supplied via the sample supplying path. The biosensor isinserted into a measuring device which includes a supporting section forsupporting detachably the biosensor, connecting terminals, and a drivingpower supply for applying a voltage to the electrode section. When thebiosensor is inserted into the supporting section of the measuringdevice, the driving power supply applies a voltage to a first electrodegroup and a second electrode group. The first group is formed of thecounter electrode and the measuring electrode, and the second group isformed of the detecting electrode and one of the counter electrode andthe measuring electrode.

In the biosensor, the detecting electrode among the counter electrode,the measuring electrode, and the detecting electrode is disposed mostdownstream along the sample supplying path, i.e., from a sample inletalong the sample flowing direction. It may be determined whether or notthe sample liquid is supplied sufficiently for the measurement dependingon whether or not respective electric currents from the first and secondelectrode groups exceed respective given thresholds.

After the electric current from the first electrode group exceeds thegiven threshold, if the current from the second group does not exceedthe given threshold within a predetermined period, it may be determinedthat the sample liquid is insufficient. In this case, the measuringdevice may output the determination to the outside.

After the electric current from the first electrode group exceeds thegiven threshold, if the current from the second group does not exceedthe given threshold within the predetermined period, an operator mayhold a measuring step in order to add the sample liquid.

In the sample supplying path of the biosensor, the detecting electrodeamong the counter electrode, the measuring electrode, and the detectingelectrode is disposed most downstream along the sample flowing directionfrom the sample inlet. An air hole for accelerating the flowing of thesample liquid is formed downstream against the detecting electrode. Ifthe electric current from the second electrode group exceeds thepredetermined threshold before the first group, and if the current fromthe first group does not exceed the threshold within a given period, itmay be determined that the sample liquid is sucked from the air hole bymistake.

A measured quantity of the substrate corresponding to electric currentdetected by the electrode section may be compensated according to alapse of time since the current from the first electrode group exceedsthe threshold until the current from the second electrode group exceedsthe threshold.

The measuring device may include a memory storing measured data whichshows correspondence between a quantity of the substrate included in thesample liquid and a current detected by the biosensor. The measuringdevice refers to the measured data, thereby determining the quantity ofthe substrate corresponding to the detected current.

After the sample liquid is supplied to the sample supplying path,reaction between the sample liquid and the reagent layer is incubatedduring a certain time, and the quantity of the substrate is thenmeasured. In this case, the incubating time may vary according to alapse of time since the current from the first electrode group exceedsthe threshold until the current from the second group exceeds thethreshold. The driving power supply may apply a voltage to the firstgroup and the second group alternately at constant intervals.

A fourth aspect of the present invention aims to provide a method ofmeasuring a quantity of a substrate. This method uses a biosensorincluding a reagent layer which reacts specifically on the substrate insample liquid. The method also uses a measuring device for measuring thequantity of the substrate included in the sample liquid from a sampleproduced by the reaction between the sample liquid and the reagentlayer. The measuring device includes a temperature measuring section formeasuring a temperature in the reaction progress between the sampleliquid and the reagent layer and a temperature compensation memory forstoring plural compensation tables of measured data. The compensationtables are prepared for each temperature range. The measuring deviceselects a compensation table according to a temperature measured by thetemperature measuring section, and calculates a compensation valueresponsive to a measured quantity of the substrate for compensation.

The biosensor may include an electrode section including a counterelectrode and a measuring electrode which are disposed on at least apart of an insulating board. The measuring device may apply a voltage tothe electrode section and detect an electric current from theelectrodes.

A fifth aspect of the present invention aims to provide a method ofmeasuring, with a measuring device, a quantity of a substrate includedin sample liquid supplied to a biosensor. The measuring device includesa temperature measuring section for measuring a temperature inside themeasuring device. The temperature measuring section detects atemperature change between a temperature measured before the measurementof the substrate quantity and a temperature at the measurement.According to the temperature change, the measuring device determineswhether the substrate quantity is to be measured or not.

If the temperature change exceeds a given threshold, the measurement maybe cancelled. A temperature prior to the measurement may be measuredintermittently.

A sixth aspect of the present invention aims to provide a method ofmeasuring a quantity of a substrate included in sample liquid with abiosensor and a measuring device. The biosensor includes an electrodesection including a counter electrode, a measuring electrode, and areagent layer for reacting on sample liquid supplied to the electrodesection which are disposed on at least part of an insulating board. Themeasuring device includes a supporting section for detachably supportingthe biosensor, connecting terminals, and a driving power supply forapplying a voltage to the electrode section. The driving power supplyapplies a voltage to the electrode section, and an electric current fromthe electrode section is detected, thereby measuring the quantity of thesubstrate included in the sample liquid. The measuring device applies afirst voltage during a first period to the electrode section of thebiosensor supported by the supporting section. After this voltageapplication during the first period, the voltage application is haltedduring a standby period. After the standby period, a second voltage isapplied to the electrode section during a second period, and the currentfrom the electrode section is measured, thereby measuring the quantityof the substrate. The first voltage is greater than the second voltage.

A seventh aspect of the present invention aims to provide a biosensorincluding two boards which are bonded to each other for forming a samplesupplying path for taking sample liquid between the boards. The sampleliquid is poured into an opening at respective ends of the boards as aninlet. The respective ends of the boards are located at different placesfrom each other in a plan view of the biosensor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a biosensor system in accordance with a first exemplaryembodiment of the present invention.

FIG. 2 is an exploded perspective view of a biosensor in accordance witha first exemplary embodiment of the present invention.

FIG. 3 shows combinations of recognizing sections of the biosensordepending on the presence of slits in accordance with the firstembodiment.

FIG. 4 shows structures of the biosensor and a measuring device inaccordance with the first embodiment.

FIG. 5 is a flowchart illustrating processes of measuring a quantity ofa substrate included in sample liquid by the biosensor and the measuringdevice.

FIG. 6 is a flowchart illustrating processes of measuring a quantity ofa substrate included in sample liquid by the biosensor and the measuringdevice.

FIG. 7 is a flowchart illustrating steps of measuring a quantity of asubstrate included in sample liquid by the biosensor and the measuringdevice.

FIG. 8 illustrates a relation between a delay time and a compensationcoefficient for compensating a measured quantity of a substrate.

FIG. 9 shows a profile at a measurement pre-process.

FIG. 10 shows a relation between a blood viscosity, a reaction time ofreactive reagent layer and blood, and a measurement sensitivity.

FIG. 11 shows a glucose concentration (mg/dl) measured by a conventionalmethod and a measurement pre-process of the present invention.

FIG. 12 shows data CA of a calibration curve.

FIG. 13 shows temperature compensation tables.

FIG. 14 shows relations between a temperature measured and measurementdispersion at each concentration of a substrate.

FIG. 15 shows a temperature change in a measuring device.

FIG. 16 is an exploded perspective view of a conventional biosensor.

FIG. 17 shows an exploded perspective view and a sectional view of abiosensor in accordance with a second exemplary embodiment of thepresent invention.

FIG. 18 is an enlarged plan view illustrating a sample supplying path ofthe biosensor shown in FIG. 17.

FIG. 19 shows an exploded view and a sectional view of another exampleof the biosensor.

FIG. 20 is an enlarged plan view illustrating a sample supplying path ofthe biosensor.

FIG. 21 illustrates a test method of sucking blood by the biosensor.

FIG. 22 illustrates another test method of sucking blood by thebiosensor.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Exemplary embodiments of the present invention will be describedhereinafter with reference to the accompanying drawings. The embodimentsdiscussed here are only examples, and the present invention is notnecessarily limited to these embodiments.

Exemplary Embodiment 1

The first embodiment will be demonstrated hereinafter with reference tothe accompanying drawings. FIG. 1 shows a biosensor system in accordancewith the first embodiment of the present invention. Biosensor system 1includes biosensor 30 and measuring device 10 having biosensor 30mounted detachably thereto. Sample liquid is dripped on sample-drippoint 30 a located at a tip of biosensor 30. A quantity of a substrateincluded in the dripped sample liquid is measured by measuring device10.

Measuring device 10 includes, for instance, supporting section 2 towhich biosensor 30 is detachably mounted and display 11 which shows ameasured quantity of the substrate included in the sample liquid drippedon sample-drip point 30 a.

To measure a quantity of a substrate included in sample liquid withbiosensor system 1, first, a user inserts biosensor 30 into measuringdevice 10. Then the user drips the sample liquid on sample-drip point 30a while measuring device 10 applies a certain voltage to electrodes ofbiosensor 30. The sample liquid dripped, upon being sucked intobiosensor 30, make a reagent layer start dissolving. Measuring device 10detects an electrical change generated between the electrodes ofbiosensor 30, then starts measuring the quantity of the substrate.

Biosensor system 1 in accordance with the first embodiment is suitableto processing human blood as a sample liquid among others, and measuringa quantity of glucose, lactic acid, cholesterol included in the humanblood as a substrate. Measuring the quantity of the substrate includedin human body fluid is very important for diagnosis and medicaltreatment for a specific physiological abnormality. In particular, adiabetic is required to monitor his glucose concentration in the bloodfrequently.

The following demonstration refers to measuring a quantity of glucoseincluded in human blood. However, biosensor system 1 in accordance withthe first embodiment can measure a quantity of lactic acid, cholesteroland other substrates by selecting an appropriate enzyme as well.

Next, components forming biosensor 30 will be described with referenceto FIG. 2, an exploded perspective view of biosensor 30. Insulatingboard 31 (hereinafter called simply “board”) is made of, e.g.,polyethylene terephthalate. On a surface of board 31, a conductivelayer, which is made of a noble metal such as gold and palladium, or anelectrically conductive substance such as carbon, is formed by screenprinting or sputtering evaporation. The conductive layer may be formedon the entire or at least a part of the surface. Reference numeral 32denotes an insulating board having air hole 33 formed at its center.Spacer 34 having a notch is disposed between boards 31 and 32, so thatboard 32 is integrated to board 31.

On board 31, the conductive layer is divided by a plurality of slitsinto counter electrode 37, measuring electrode 38, and detectingelectrode 39. In detail, the conductive layer is divided by thefollowing slits: substantially arc-shaped slit 40 formed on counterelectrode 37; slits 41 a and 41 c formed vertically to a side of board31; slits, 41 b, 41 d, and 41 f and V-shaped slit 41 e. The slits formcounter electrode 37, measuring electrode 38 and detecting electrode 39.Each electrode may can be formed on at least a part of board 31.Measuring device 10 may connected to the electrodes with lead wires.

Spacer 34 is placed for covering counter electrode 37, measuringelectrode 38, and detecting electrode 39 on board 31. The notch shapedin a rectangular provided at a center in a front section of spacer 34forms sample supplying path 35. The sample liquid is dripped to inlet 30a of sample supplying path 35. The sample liquid dripped to inlet 30 ais sucked by capillary phenomenon in an approximately horizontaldirection (along arrow AR in FIG. 2) toward air hole 33.

Reference numeral 36 denotes a reagent layer formed by applying reagent,which contains enzymes, electron acceptors, amino acid, sugar alcoholand the like, to portions of counter electrode 37, measuring electrode38 and detecting electrode 39, the portions which are exposed from thenotch of spacer 34.

The enzymes may employ the following materials: glucose oxidase, lactateoxidase, cholesterol oxidase, cholesterol estrase, uricase, ascorbateacid oxidase, bilirubin oxidase, glucose dehydrogenase, lactatedehydrogenase.

The electron acceptor preferably employs ferricyanide kalium, however,may employ p-benzoquinone and its derivatives, phenacine methorsulphate, methylene blue, and pherocane and its derivatives.

In the biosensor system in accordance with the first embodiment, glucoseoxidase is used as oxidoreductase retained in reagent layer 36, andferricyanide kalium is used as the electron acceptor in order to measurethe glucose concentration in human blood.

The oxidoreductase and the electron acceptor dissolve in the sampleliquid (human blood in this embodiment) which is sucked into the samplesupplying path, and then the glucose, a substrate in the sample liquid,reacts with the oxidoreductase and the electron acceptor, and the enzymereaction progresses. Then the electron acceptor is reduced, thusproducing ferrocyanide (ferricyanide kalium in this embodiment). Afterthe reaction, the reduced electron acceptor, upon being oxidizedelectrochemically, generates a current from which the glucoseconcentration is measured. This series of reactions progress mainly inan area covering slits 40, 41 e and detecting electrode 39. The currentproduced by the electrochemical change is read out through measuringelectrode 38 and detecting electrode 39.

Reference numeral 42 denotes a recognizing section for recognizing, withmeasuring device 10, a type of biosensor 30 and a difference in outputcharacteristics among production lots. Slits 41 g and 41 h are combinedto portions of counter electrode 37 and detecting electrode 39corresponding to recognizing section 42. The slits enables measuringdevice 10 to recognize the differences in output characteristicselectrically.

FIG. 3 shows combinations of slits depending on the presence of slits 41g, 41 h in recognizing section 42 of biosensor 30. FIG. 3 illustratesseven types of combinations. For instance, FIG. 3( a) shows recognizingsection 42 of biosensor 30 for measuring cholesterol. In this case,slits 41 g and 41 h are not formed.

FIGS. 3( b), 3(c), and 3(d) illustrate recognizing section 42 ofbiosensor 30 for measuring lactic acid. In FIG. 3( b), slit 41 h isprovided only in counter electrode 37, thereby forming compensatingsection 43. In FIG. 3( c), slit 41 g is provided only to detectingelectrode 39, thereby forming compensating section 44. In FIG. 3( d),slits 41 h and 41 g are provided to counter electrode 37 and detectingelectrode 39, respectively, thereby forming compensating section 43 and44, respectively. Further, FIGS. 3( e), 3(f), and 3(g) illustraterecognizing section 42 of biosensor 30 for measuring glucose. In FIG.3(e), slit 41 g is provided only to detecting electrode 39, and slit 41d is formed up to slit 41 g. And thus compensating section 44 isintegrally formed with measuring electrode 38. In FIG. 3( f), slit 41 his added to the section in FIG. 3( e), thereby forming compensatingsection 43. In FIG. 3( g), slit 41 f is formed up to slit 41 h shown inFIG. 3( f). Thus, correcting sections 43 and 44 are integrally formedwith measuring electrode 38.

As such, a conductive area between the electrodes can be varieddepending on patterns of the slits in recognizing section 42. Thisenables measuring device 10 to recognize the differences in outputcharacteristics (concentrations of glucose, cholesterol, lactic acid) ofbiosensor 30 and errors depending on production lots. Data and a controlprogram, since being changed appropriately to the substrate according tothe recognition, enables the device to be expected in exact measurement.This allows a user not to input compensating data using a compensatingchip, and prevents the user from incorrectly handling the device. Thisembodiment discloses the biosensor having three electrodes. However, anumber of electrodes may change, and a biosensor may have at least apair of electrodes. The patterns of the slits other than those shown inFIG. 3 may be formed.

Next, a structure of measuring device 10 will be explained in detail.FIG. 4 shows structures of biosensor 30 (top view) and measuring device10. In biosensor 30, counter electrode 37, measuring electrode 38 anddetecting electrode 39 are arranged along a flowing direction of asample from sample-drip point 30 a where detecting electrode 39 isplaced most downstream. Counter electrode 37 may be exchanged betweenmeasuring electrode 38 in the arrangement order. Measuring electrode 38and detecting electrode 39 are spaced at a given distance by slits 41 cand 41 e. Thus, the device can determine, from an electric currentchanging according to an electrical change of the substrate, whetherenough quantity of the sample liquid is sucked securely or not.

In measuring device 10, reference numerals 12, 13, 14, 15, 16 and 17denote connectors connected to areas A, B, C, D, E and F, respectively,which are produced by dividing recognizing section 42 of biosensor 30into six areas. The six areas are grouped such that the groupscorrespond to slits 41 d, 41 f and slits 41 g, 41 h. Area A correspondsto measuring electrode 38, area C corresponds to detecting electrode 39,and area E corresponds to measuring electrode 38. Area A is integrallyformed with area B, and areas D and F correspond to compensatingsections 43 and 44 shown in FIG. 3, respectively. Switches 18, 19, 20,21 and 22 are provided between respective connectors 13, 14, 15, 16, 17and a grounding (meaning a constant voltage, not necessarily “0”V. Thisdefinition is applicable to this description hereinafter.) Voltage to beapplied to respective electrodes can be controlled at the grounding.Connectors 13, 14, 15, 16 and 17 are connected in parallel to thegrounding. Switches 18 to 22, upon being turned on and off undercontrol, select a necessary connector out of connectors 13 to 17 whichis used for the measurement.

Reference numeral 23 denotes a current/voltage converter connected toconnector 12, for converting a current flowing between measuringelectrode 38 and other electrodes into a voltage. Reference numeral 24denotes an A/D converter connected to current/voltage converter 23, forconverting a voltage supplied from circuit 23 into a pulse. Referencenumeral 25 denotes a CPU for controlling to turn on and off the switchesand calculating a content of the substrate included in the sample liquidbased on the pulse supplied from A/D converter 24. Reference numeral 11denotes an LCD for displaying measured data calculated by CPU 25.Reference numerals 26 and 28 denote temperature measuring sections formeasuring temperatures inside measuring device 10. Temperature measuringsections 26 and 28 are connected in parallel to each other betweenconnector 12 and current/voltage converter 23.

In measuring device 10 in accordance with the first embodiment, voltages(mV) converted from the currents flowing between the electrodes ofbiosensor 30 are used for detecting changes of the currents. In otherwords, the voltages indicate the currents flowing between theelectrodes.

An operations of biosensor 30 and measuring device 10 will bedemonstrated with reference to FIG. 5 through FIG. 7, for measuring acontent of a substrate in sample liquid by a method with biosensor 30according to this embodiment.

First, it is determined whether or not biosensor 30 is properly insertedinto supporting section 2 of measuring device 10 (Step S1).Specifically, this is determined with a switch (not shown) in aconnector shown in FIG. 4. If biosensor 30 is properly inserted (stepS1: Yes), conductivity between areas A and B is tested (step S2). Asshown in FIG. 3, measuring electrode 38 has no slit formed therein forinsulating one electrode itself such as slits 41 h and 41 g. Inmeasuring electrode 38, areas A and B are connected to connectors 12 and13, respectively. Areas A and B thus become conductive to each otherwithout failure when biosensor 30 is inserted into measuring device 10in a direction (a predetermined direction) such that a conductive layerof biosensor 30 is oriented normally.

Therefore, conductivity between areas A and B is tested by turning onswitch 18, so that the front and back sides of biosensor 30 can bedetermined. If the conductivity between areas A and B is not detected(step S2: No), it is determined that biosensor 30 is inserted front-sideback (reversely). Then the measuring process terminates due to an errorof detecting the front and back sides (step S3). The error, when beingdetected, is preferably displayed on display 11, or noticed as an alarmsound from a speaker. These preparations prevent the user easily fromdripping blood to biosensor 30 by mistake while biosensor 30 is insertedfront-side back.

When the conductivity between areas A and B is detected (step S2: Yes),it is determined whether or not voltages detected between area A andarea C and between area A and area E are greater than 5 mV (step S4).Switches 19 and 21 are simultaneously turned on, thereby allowing areasC and E to be considered to be electrically unified. Then a voltage isdetected between area A and area C or E for determining whetherbiosensor 30 inserted in step 1 is an used one or not. This isdetermined since a reaction between reagent layer 36 and glucose in theblood has progressed to probably enlarge the detected voltage ifbiosensor 30 is the used one.

If it is determined that the voltage detected between area A and areas Cis greater than 5 mV (step S4, Yes), it is recognized that biosensor 30which is used is inserted, and the measuring process terminates due toan error of an used sensor (step S5). If being detected, the error ofused sensor is preferably displayed on display 11, or noticed to a useras an alarm sound from a speaker. This prevents the user easily fromdripping blood to biosensor 30 by mistake while used biosensor 30 isinserted. Next, when the voltage detected between area A and areas C, Eis not greater than 5 mV (step S4: No), the patterns of the slits isrecognized by recognizing section 42 of biosensor 30 which is detectedto be inserted at step S1. According to the recognizing result, CPU 25changes data and a program into appropriate ones for outputcharacteristics of the sensor (steps S6 to S10). In the firstembodiment, three patterns of the slits are available, as shown in FIGS.3( e), 3(f), and 3(g), for a blood-sugar-level sensor which measures aglucose concentration. Specifically, first, conductivity between areas Aand D is tested (step S6). Switch 20 is turned on, and then theconductivity between areas A and D is tested, so that it may bedetermined whether or not biosensor 30 is proper to measure a bloodsugar level and not proper to measure a quantity of lactic acid orcholesterol.

If the conductivity between areas A and D is not detected (step S6: No),it is determined that biosensor 30 is incompatible with theblood-sugar-level sensor. Then the measuring process terminates (stepS7), and display 11 shows an error message, or a speaker sounds an alarmfor the user. These prevent the user from recognizing a measurement as aglucose concentration by mistake.

If the conductivity between areas A and D is detected (step S6: Yes),the conductivity between areas A and F is tested (step 8). Switch 22 isturned on. Then the conductivity between areas A and F is tested, sothat the device can recognize differences in output characteristics dueto production lots of biosensors 30 proper to blood-sugar-level sensors.CPU 25 automatically changes data and programs to which outputcharacteristics corresponding to production lots have been reflected.Thus the user does not need a compensating chip. As a result, thebiosensor and the measuring device can be handled more easily, and ahigher accuracy of measurement can be expected.

If conductivity between areas A and F is detected (step S8: Yes),biosensor 30 is defined as a type shown in FIG. 3( g), and result I isstored in a memory (not shown) (step S9). If the conductivity betweenareas A and F is not detected (step S8: No), biosensor 30 is defined asa type shown in FIG. 3( e) or FIG. 3( f), and result II is stored in thememory (not shown) (step S10).

After the type of biosensor 30 is recognized, it is determined againwhether the voltage detected between area A and areas C, E is greaterthan 5 mV or not (step S11). Switches 19, 21 are simultaneously turnedon for detecting a current between area A and areas C, E. Then it isdetermined whether or not a user drips the sample liquid on biosensor 30before measuring device 10 is ready for measurement. This process notonly prevents positively the user from using used biosensor 30, but alsodetects that the sample liquid has been dripped by the user before themeasurement is available.

If the voltage detected between area A and areas C, E is greater than 5mV (step S11: Yes), it is determined, as a drip error, that the sampleliquid is dripped before the measurement is prepared. When beingdetected, the drip error is preferably displayed on display 11, notifiedto a user with an alarm sound from a speaker, or displayed with LEDs(not shown) to give the user an alarm. The user can positively avoid afailure in operation by these operations, and a high accuracy ofmeasurement can be expected.

If the voltage detected between area A and areas C, E is not greaterthan 5 mV (step S11: No), it is determined that the sample liquid is notdripped before the measurement is prepared. Then a completion of thepreparation is notified to the user with LEDs (step S13). When beingdetected, the error is preferably displayed on display 11, notified tothe user with an alarm sound from a speaker, or displayed with LEDs.Receiving this notice, the user takes blood as sample liquid from hisbody by himself and drips it to sample-drip point 30 a of biosensor 30inserted to measuring device 10.

Next, it is determined whether or not enough quantity of the sampleliquid is sucked through the sample supplying path from point 30 a(steps S14 to S20). In biosensor 30, counter electrode 37, measuringelectrode 38, and detecting electrode 39 are arranged along samplesupplying path 35 from sample-drip point 30 a toward a downstream of thesample liquid flow. Detecting electrode 39 is placed most downstream.Either one of a group consisting of counter electrode 37 and measuringelectrode 38, or another group consisting of measuring electrode 38 anddetecting electrode 39 is selected at a given interval. A voltage isapplied to a selected group, so that it is determined whether or not thesample liquid is supplied in a quantity enough for measurement. In aconventional manner, a current change only between measuring electrode38 and detecting electrode 39 is recognized. In the conventional manner,it is very difficult to identify a cause why the measurement does notstart even though enough quantity of the sample liquid is supplied tothe sample supplying path, or since the quantity is less than enoughquantity for starting the measurement.

Specifically, for the group of counter electrode 37 and measuringelectrode 38, switch 19 is turned off, and switch 21 is turned on forgenerating a voltage between areas A and E. For the group of measuringelectrode 38 and detecting electrode 39, switch 19 is turned on, andswitch 21 is turned off for generating a voltage between areas A and C.As such, switches 19 and 21 are on-off controlled, thereby selecting andswitching either one of the groups easily. For easy description,hereinafter, generating the voltage between counter electrode 37 andmeasuring electrode 38 is referred to as generating a voltage betweenareas A and E. Also generating a voltage between measuring electrode 38and detecting electrode 39 is referred to as generating a voltagebetween areas A and C.

Further in this embodiment, as an example, a pair of areas A and E and apair of areas A and C are switched every 0.2 seconds, and 0.2V isapplied to each pair. It is determined whether or not respectivevoltages measured between areas A and E and between areas A and Creaches 10 mV (a given threshold). These numbers may be changedresponsive to a type of biosensors.

Back to the flowchart shown in FIG. 6, the operations of the biosensorand the measuring device will be further described hereinafter. First, avoltage of 0.2V is produced between areas A and E which are located atthe upstream portion of the sample supplying path, and it is determinedwhether or not the voltage measured between areas A and E exceeds 10 mV(step S14). If the voltage measured does not exceed 10 mV (step S14:No), a voltage of 0.2V is applied between areas A and C locateddownstream of the path. Then it is determined whether the voltagemeasured between areas A and C exceeds 10 mV or not (step S15).

If the voltage measured between areas A and C does not exceed 10 mV(step S15: No), it is determined whether or not 3 minutes have passedsince the voltage was produced between areas A and E in step S14 (stepS16). If the 3 minutes has not passed (step S16: No), the processes fromstep S14 and onward are repeated. If respective voltages between areas Aand E and between areas A and C do not reach 10 mV for 3 minutes (stepS16: Yes), the measuring process terminates.

If the voltage between areas A and E is determined to reach 10 mV (stepS14: Yes), it is determined whether or not the voltage between areas Aand C reaches 10 mV (step S17). If the voltage between areas A and Cdoes not reach 10 mV (step S17: No), it is determined whether or not 10seconds (a given period) have passed since the voltage between areas Aand E was determined to reach 10 mV (step S18). If the 10 seconds hasnot passed, the processes in steps S17 and S18 are repeated. While the10 seconds passes, the measuring process temporarily halts until thevoltage measured between areas A and C reaches 10 mV (step S18; No). Inthis case, the sample liquid dripped is probably insufficient, it ispreferable to display the error message on display 11, or sound an alarmto a user from a speaker so that the user may understand that the sampleliquid should be added. If the voltage measured between areas A and Cdoes not reach 10 mV even after 10 seconds has passed (step S18: Yes),the measuring process terminates due to an error of specimeninsufficient (step S19).

While the 10 second passes since the voltage between areas A and E wasdetermined to reach 10 mV in step S14, if a user adds the sample liquid,a final measurement accuracy is lowered. This was found by inventors.Specifically, while the user adds the sample liquid, the substrate inthe sample liquid originally dripped has reacted on the enzyme includedin reagent layer 36 and enzyme reaction has progressed. Thus a reducedsubstance has been produced before the measurement starts. After theadded sample liquid reaches between areas A and C, the quantity of thesubstrate is possibly measured. In this case, the reduced substancealready produced influences this measurement, i.e., makes the voltageapparently greater. In other words, as a time since the voltage betweenareas A and E is determined to reach 10 mV in step S14 becomes longer,the measurement is influenced more by the reduced form.

In order to eliminate a measurement error caused by adding the sampleliquid, a quantity of the substrate is compensated responsive to ameasured voltage in measuring device 10 in accordance with thisembodiment. The compensation depends on the lapse of time (delay time)since the voltage between areas A and E was determined to reach 10 mV instep S14 until the voltage between areas A and C is determined to reach10 mV in step S17.

FIG. 8 is a sensitivity compensation table illustrating a relationbetween the delay time and a compensation coefficient for the measuredquantity of the substrate. The vertical axis represents the compensationcoefficient, and the horizontal axis represents the delay time. Forinstance, if the delay time is 5 seconds, the measured quantity iscompensated by 10% lower. As a result, 90% of the measured quantitybecomes a compensated quantity. This kind of the sensitivitycompensation table is stored in a memory (not shown). of measuringdevice 10, and this table is referred when a final quantity of thesubstrate is calculated.

In biosensor 30 shown in FIG. 2, counter electrode 37 is formed suchthat slit 41 f extends toward slit 41 c and connects with slit 41 b.Then a drip position error caused through dripping the sample liquid toair hole 33 by mistake, can be detected. In the flowchart shown in FIG.6, if the voltage measured between areas A and C is determined to excess10 mV not the voltage between areas A and E (step S15: Yes), it isdetermined, in 0.2 seconds after the determination, whether or not thevoltage between areas A and E reaches 10 mV (step S20). If the voltagebetween areas A and E does not excess 10 mV, it is determined that thesample liquid has been dripped to an incorrect position, and themeasuring process terminates (step S50).

If the sample liquid is normally dripped on sample-drip point 30 a, theliquid is sucked along sample supplying path 35 to air hole 33 and thenmoistens counter electrode 37, measuring electrode 38 and detectingelectrode 39 in this order. However, if the voltage measured onlybetween areas A and C changes largely, a user has probably dripped thesample liquid to air hole 33 incorrectly. In this case, it is determinedthat an exact measurement is not expected, and the measuring processcompulsorily terminates due to an error of dripping at a incorrectposition. This can avoid a measurement error due to an incorrectoperation by the user.

If the voltage measured between areas A and C is determined to reach 10mV (step S17: Yes), or if the voltage measured between areas A and E isdetermined to reach 10 mV (step S20: Yes), enough quantity of the sampleliquid is determined to be dripped. Then a pre-process for measuring thequantity of the substrate starts, and a timer (not shown) of measuringdevice 10 counts time (step S21).

Next, conductivity between areas A and F is tested (step S22). Switch 22is turned on, and the conductivity is tested between areas A and F. Ifthe conductivity is detected (step S22: Yes), it is determined whetherresult I identifying a type of biosensor 30 is stored in the memory instep S9 or not (step S23). If result I is stored (step S23: Yes), it isdetermined that the type of biosensor 30 is that shown in FIG. 3( g).Calibration curve data is prepared using voltages measured when thereduced electron acceptor is oxidized electrochemically. Thencalibration curve F7 is prepared as the calibration curve data forspecifying a concentration of the glucose in the sample liquid (stepS24).

On the other hand, when result II is stored (step S23: No), it isdetermined that the type of biosensor 30 is that shown in FIG. 3( e),and calibration curve F5 is prepared as the calibration curve data (stepS25). If the conductivity is not detected between areas A and F (stepS22: No), it is determined that the type of biosensor 30 is that shownin FIG. 3( f), and calibration curve F6 is prepared as the calibrationcurve data (step S26).

As discussed above, a difference in output characteristics of biosensor30 is automatically recognized responsive to the slits in recognizingsection 42 of biosensor 30. The calibration curve data appropriate tothe output characteristics is then automatically selected and set.Therefore, a user does not need a compensating chip, and CPU 25 switchesautomatically the calibration curve data to which the outputcharacteristics depending on production lots are reflected. As a result,an incorrect measurement using user's incorrect data can be avoided, anda highly accurate measurement can be expected.

After the calibration curve is prepared in steps S24 to S26, themeasuring pre-process starts (step S27-S29). The pre-process will bedemonstrated with reference to FIG. 9, which illustrates a profile ofthe pre-process in accordance with the first embodiment.

In the profile shown in FIG. 9, the pre-process starts at time t0.Specifically, time t0 is the time when the timer (not shown) ofmeasuring device 10 starts counting time. The profile of the pre-processincludes three consecutive periods, for instance, a first voltage periodt0-t1, a standby period t1-t2, and a second voltage period t2-t3.

During the first voltage period, voltage V1 is applied to areas A, C andE, to have the enzyme reaction progress. This increases a voltagemeasured by oxidizing ferrocyanide electrochemically similar to anexponential function. Next, during the standby period, voltage V1applied during the first voltage period is set at zero, and thus theferrocyanide is not oxidized electrochemically, but the enzyme reactionkeeps progressing. The ferrocyanide is thus accumulated. During thesecond voltage period, voltage V2 is applied to areas A, C and E tooxidize the ferrocyanide accumulated during the standby period all atonce. Then a quantity of discharged electron increases, and a highresponse current is thus observed at time t2. A current reaching thehigh response current decreases, as time passes, into a stable value i3at time t3. In the pre-process, switches 19 and 21 are simultaneouslyturned on in measuring device 10, so that a voltage is applied tocounter electrode 37 and detecting electrode 39 as one unit.

Recently, shortening a measurement time has been desired to upgrade aperformance of the biosensor. When a quantity of a substrate is measuredwith a biosensor, a viscosity of the sample liquid critically influencesmeasurement accuracy. This was found by the inventor. In particular,when human blood is measured as the sample liquid, blood with highviscosity (high Hct) lowers measurement sensitivity, and blood with lowviscosity (low Hct) increases the measurement sensitivity. Thisphenomenon derives from a dissolving speed of a reagent layer in theblood, i.e., slow dissolution in high Hct and quick dissolution in lowHct. Thus the viscosity influences the measurement sensitivity of thebiosensor.

FIG. 10 shows a relation between a blood viscosity, a reaction time ofreactive reagent layer on the blood, and a measurement sensitivity. Datashown in FIG. 10 is measured by a conventional method, which applies avoltage within a period corresponding to the second voltage period shownin FIG. 9 and measures the voltage. As shown in FIG. 10, influence dueto differences in viscosity (Hct in the case of blood) to measurementsensitivity increases at a shorter reaction time. Great difference isobserved between the high Hct and the low Hct particularly at a reactiontime around 5 sec.

Therefore, the conventional method tends to reveal a measurement errorobviously due to blood viscosity.

During the first voltage period of the pre-process, reaction productsproduced at an initial stage of dissolving reagent layer 36 is thuscompulsorily consumed by applying voltage V1. During the first voltageperiod, since the low Hct has a higher speed in enzyme reaction than thehigh Hct, greater reaction products are produced in the low Hct and thusgreater reaction products are consumed. However, if a voltage is appliedfor too long period, reaction products are consumed too much, andresponsivity of a voltage detected in the second voltage period mayprobably decline. Therefore, an effective first voltage period t1-t0 maybe 3 to 13 seconds. The voltage to be applied may be further increased,so that a voltage application time is preferably 2 to 10 seconds.Voltage V1 may range preferably from 0.1 to 0.8V.

Next, during the standby period, the enzyme reaction progresses again,and the reaction products in the low Hct blood, the reaction productswhich have been consumed in the first voltage period, are quicklyrecovered and accumulated in approximately the same quantity as those inthe high Hct blood. Too long a standby period or too short a standbyperiod influences the final measurement sensitivity in a different way.

If the standby period is too short, a response value i3 measured at timet3 becomes too low, and a measurement error becomes great. If thestandby period is too long, a difference in enzyme reaction speedbetween the low Hct blood and the high Hct blood probably becomesgreater. The standby period is determined so that the difference inenzyme reaction speeds may not become greater. As a result, the standbyperiod t2-t1 is 1 to 10 seconds and preferably 2 to 10 seconds.

During the second voltage period, voltage V2 starts being applied attime t2. And just after time t2, the voltage is not stable and requiresa time to be stable. A voltage similar to that during the first voltageperiod is not necessarily applied, and a lower voltage than voltage V1is preferably applied. The lower voltage may be low enough to oxidizeferrocyanide kalium. The second voltage period t3-t2 is thus preferably2 to 10 seconds. Voltage V2 is preferably 0.05 to 0.6V. Finally, valuei3 measured between areas A, C and area E at time t3 is read out, andthe quantity of the substrate (glucose) in the sample liquid iscalculated.

The set time discussed above is particularly suitable for a quantitymeasuring with the biosensor including electrodes made of noble metalsuch as palladium. A reagent is not limited to glucose oxidase and/orglucose dehydrogenase and ferricyanide kalium, but includes amino acid,sugar alcohol. The set time is also suitable to a biosensor includingorganic acid.

After the sample liquid is supplied to sample supplying path 35, thereaction of reagent layer 36 in the sample liquid is incubated in acertain period before the quantity of the substrate is measured. Theincubate period may change depending on the laps of time since thevoltage measured between areas A and E exceeds a threshold (10 mV) instep S14 until the voltage between areas A and C exceeds the threshold(10 mV) in step S17.

FIG. 11 shows glucose concentrations (mg/dl) measured by theconventional method and the measuring pre-process discussed above forthree types of blood having contents of hematocrit (Hct) of 25%, 45% and65%. Reference mark R in FIG. 11 denotes the measurement result by thepre-process. The other two results were measured by the conventionalmethod with 15 seconds and 30 seconds of the reaction time. Thepre-process was performed under the following condition: the firstvoltage period was 6 seconds; voltage V1 was 0.5V; the standby periodwas 6 seconds; the second voltage period was 3 seconds; and voltage V2was 0.2V. As compared with a measurement for: the Hct was 45%; and theglucose concentration was 100 mg/dl, an actual measurement proved thatlow Hct (25%) blood and high Hct (65%) blood produce greater dispersionin the measurement, and the response values of low Hct disperse in ahigher range and those of high Hct disperse in a lower range. Further,the dispersion becomes greater at a shorter reaction time. At a reactiontime of 15 seconds, the dispersion is produced by 10% higher (low Hct of25%) and by 10% lower (high Hct of 65%). At a reaction time of 30seconds, the dispersion is produced by 5% higher (low Hct of 25%) and by5% lower (high Hct of 65%). In this pre-process, the dispersion isproduced by 3% higher (low Hct of 25%) and by 3% lower (high Hct of65%). At a reaction time of 15 seconds, FIG. 11 teaches that thepre-process can reduce the dispersion due to the types of Hct while thereaction time is the same as that in the conventional method.

Back to FIG. 7 again, the description of the measuring process continueshereinafter. The measuring pre-process starts, and 0.5V is appliedbetween areas A and C, and between areas A and E for 6 seconds in thefirst voltage period (step S27). After the first voltage period, thestandby period is taken for 6 sec., and the voltage applied is cancelledin the standby period (step S28). After the standby period, the secondvoltage period starts, and 0.2V is applied between areas A and C, andbetween areas A and E for 3 sec. (step S29). Then value i3 is read out(step 30).

After value i3 is read out in step S30, temperature measuring sections26 and 28 and switches 27, 29 disposed in measuring device 10 arecontrolled to measure a temperature in measuring device 10 (step S31).Specifically, switch 27 is turned on, and measuring section 26 measuresthe temperature (step S31). Then switch 27 is turned off, switch 29 isturned on, and measuring section 28 measures the temperature (step S32).

The two temperatures measured by temperature measuring section 26 and 28are compared with each other, and it is determined whether or not thedifference between the two temperatures ranges within a given threshold(step S33). If the difference is out of the threshold, the measuringprocess terminates due to a failure of either one of measuring section26 or 28 (step S33: No). As such, plural temperature-measuring sections(26, 28) are disposed in measuring device 10, and their measuringresults are compared, so that a failure can be detected exactly andeasily. This can avoid a measurement error caused by a measurement at anirregular temperature. The temperatures are measured just after thevalue has been read out in step S30; however, the temperatures may bemeasured, for instance, when the pre-process starts in step S21. If thedifference between the two temperatures measured ranges within the giventhreshold (step S33: Yes), the temperatures are temporarily stored in amemory (not shown). At this time, the temperature measured by either oneof sections 26 or 28 may be selected and stored, and the average of thetwo temperatures may be stored. Then a calibration curve, which shouldrefer to value i3 measured in step S30, is specified (step S34). Thecalibration curves prepared in steps S24, S25 and S26 are referred. Ifbiosensor 30 corresponds to step S24, calibration curve F7 is referred(step S35). In the same manner, if biosensor 30 corresponds to step S25,calibration curve F5 is referred (step S36). If biosensor 30 correspondsto step S26, calibration curve F6 is referred (step S37).

FIG. 12 shows calibration curve data CA measured in steps S34, S35 andS36. In data CA, a voltage measured in step S30 and a concentration(mg/dl) of a substrate included in sample liquid are determineddepending on each output characteristic F1 to F7 of biosensor 30. Forinstance, if a measured voltage is 25 mV, and the biosensor correspondsto calibration curve F5, a substrate concentration of 14 (mg/dl) isstored in the memory.

Next, a concentration of the substrate selected in step S35, S36 or S37is compensated by a compensation coefficient corresponding to the delaytime which has been found in steps S14 and S17 and stored in the memory(step S38). Specifically, the concentration is compensated by thefollowing equation (1):D1=(concentration of substrate)×[{100−(sensitivity compensationcoefficient)}/100]where D1 is a compensated concentration of the substrate. Thiscompensation eliminates a measurement error due to adding sample liquidby a user.

Next, the concentration compensated in step S38 is compensated accordingto the temperatures measured in steps S31 to S33 (step S39). Thetemperature stored in the memory in step S33 is read out, and atemperature compensation table shown in FIG. 13 is referred, therebydetermining a temperature compensation coefficient to be applied toconcentration D1.

FIG. 13 shows temperature compensation tables. Compensation table T10 isused for the temperature of 10° C. In the same manner, table T15 is forthe temperature of 15° C., and table T20 is for the temperature of 20°C. The compensation tables specifies a relation between substrateconcentration D1 in the sample liquid and a temperature compensationcoefficient is specified. The temperature compensation coefficient isdetermined based on a concentration at 25° C. as a reference, and showsa coefficient for compensation with respect to the concentration.Specifically, the compensation for temperature is performed according tothe following equation (2):D2=D1×(100−Co)/100where D2 is a compensated concentration, D1 is the concentrationcalculated in step S38, and Co is the temperature compensationcoefficient specified by referring to the temperature compensationtable.

The inventors found experimentally that measurement accuracy wasinfluenced by a combination of a measured temperature and aconcentration of a substrate. The influence will be describedhereinafter. FIG. 14 shows relations between the measured temperatureand measurement dispersion (bias) at each concentration of glucose. Themeasurement dispersion in FIG. 14 is defined by a coefficient of achange of a concentration of glucose measured at 25° C. according to achange of the measured temperature. FIG. 14( a) shows a relation betweenthe dispersion and the measured temperature in the case of glucoseconcentration of 50 mg/dl at 25° C. Similarly, FIG. 14( b) shows therelation for the glucose concentration of 100 mg/dl and the temperatureof 25° C. FIG. 14( c) shows the relation for the glucose concentrationof 200 mg/dl and the temperature of 25° C. FIG. 14( d) shows therelation for the glucose concentration of 300 mg/dl and the temperatureof 25° C. FIG. 14( e) shows the relation for the glucose concentrationof 420 mg/dl and the temperature of 25° C. FIG. 14( f) shows therelation for the glucose concentration 550 mg/dl and the temperature of25° C.

These experimental data point out the following two tendencies. First,for the same glucose concentration, the measuring dispersion increasesas a difference between a measured temperature and reference temperature25° C. becomes greater. In detail, the dispersion increases in anegative direction as a measured temperature decreases from thereference temperature, and the dispersion increases in a positivedirection as a measured temperature rises from the referencetemperature. Second, the dispersion converges at the glucoseconcentration of 300 mg/dl, which seems a boundary, even though theglucose concentration increases. Specifically, FIG. 14( a) indicates thedispersion of approximately 28% at 40° C., FIG. 14( c) indicatesapproximately 50%, FIG. 14( d) indicates approximately 60%, and FIG. 14(f) indicates approximately 50%. A similar tendency is found in a lowtemperature range such as a measured temperature of 10° C.

This tendency is reflected to the tables shown in FIG. 13. First, themeasuring dispersion increases as a difference between a measuredtemperature and reference temperature of 25° C. becomes greater for thesame glucose concentration. Second, the dispersion starts converging atthe glucose concentration of 300 mg/dl as a boundary even though theglucose concentration increases. These two aspects are taken intoconsideration for preparing the tables. The measurement accuracy isremarkably improved by compensating a concentration referring to thetemperature compensation table, in which combinations of measuredtemperatures and concentrations of the substrate are well considered,rather than compensating a concentration only based on a measuredtemperature.

In an operable temperature range of biosensor 30 (10° C. to 40° C. inthis embodiment), a temperature compensation table for every 1° C. maybe prepared, or the table for every given temperature range (e.g. 5°C.). If a temperature at a middle of the given temperature range isdetected, a temperature compensation coefficient may be calculated by alinear interpolation with a temperature compensation table including thedetected temperature.

Back to the flowchart in FIG. 7, concentration D2, which has undergonethe temperature compensation discussed above, is output on display 11 ofmeasuring device 10 as a final concentration of the substrate (stepS40). As discussed above, the time when the sample liquid is added, themeasured temperature, and the combination of the measured temperatureand the concentration are considered as influence factors to themeasurement. A viscosity (Hct) of sample liquid is also considered as aninfluence factor. Those factors are taken into consideration when thequantity of a substrate is measured. As a result, the measurementaccuracy is remarkably improved from the measurement by a conventionalmethod.

The following method can be introduced in order to further decrease ameasurement error due to temperature.

Before biosensor 30 is inserted into measuring device 10, thetemperature is measured successively and stored. After biosensor 30 isinserted, temperatures measured in steps S31 and S32 are compared withthe stored ones. If large differences between the stored temperaturesand the measured ones are found, the measuring process may compulsorilyterminates due to a significant temperature change which influences ameasurement error.

A portable biosensor system in accordance with this first embodiment,being carried easily, is exposed in various temperature changesdepending on the outside environment. For instance, the biosensor systemmay be influenced by a temperature of a user's hand, or a sharp changein temperature when a user moves from outside to indoors. The sharptemperature-change can be expected, it takes reasonable time formeasuring device 10 to be stabilized in its temperature change.

FIG. 15 shows temperature changes in measuring device 10. A temperaturechange in device 10 moving from a place at a temperature of 10° C. toanother place at a temperature of that of 25° C. is shown in FIG. 15. Atemperature change in device 10 moving from a place at a temperature of40° C. to a place of a temperature of 25 C. is also shown in FIG. 15.FIG. 15 shows that it takes approximately 30 minutes to stabilize thetemperature changes in an ambient temperature ranging from 10 to 40° C.If the temperature compensation is carried while the temperaturechanges, an exact temperature compensation may not be expected.

Therefore, if a great difference between the temperature stored inadvance and the temperatures measured in steps S31 and S32, themeasuring process may compulsorily terminate due to the temperaturechange which may influence a measuring error. This further improves theaccuracy of temperature compensation in measuring device 10. Atemperature may be measured before biosensor 30 is inserted intomeasuring device 10 at given intervals, e.g., 5-minute interval, orsuccessively. Based on the magnitude of temperature change, themeasuring process may be cancelled although a user tries to carry itout.

Exemplary Embodiment 2

The biosensor in accordance with the second exemplary embodiment will bedemonstrated hereinafter. In this embodiment, an enzyme sensor isdescribed. The sensor employs an enzyme as a molecule recognizingelement which specifically reacts on a specific material contained insample liquid.

An incorrect operation by a user influences a measuring accuracy. Thusthe second embodiment discusses this problem. In particular, a userfails to drip sample liquid to an inlet of a sample supplying path, andthe sample liquid attaches to a surrounding areas of the inlet. As aresult, the sample supplying path cannot carry the sample liquid. Suchkind of incorrect operations by a user may affect a measurementaccuracy, and the ways how to avoid those mis-operations aredemonstrated in this embodiment.

According to a structure shown in FIG. 16 or FIG. 2, at the inlet, towhich sample liquid is supplied, of the sample supplying path, aninsulating board and a cover forming the path have respective ends ofthe same shape at the same location in a plan view. Therefore, a samplesupplying angle becomes small. Or when the sample liquid attaches to arear side (a side having no electrode formed thereon) of the insulatingboard by mistake, this sample liquid attached to the rear side mayprevents the user from again supplying the sample liquid. As a result,the sample liquid is not supplied well, which causes a failure inmeasurement or a measurement error.

A biosensor which can accept the sample liquid exactly and easily willbe specifically described hereinafter. FIG. 17( a) is an explodedperspective view of the biosensor in accordance with the secondembodiment. FIG. 17( b) is a cross section at a center of the samplesupplying path in the longitudinal direction of the biosensor. In FIG.17, measuring electrode 52, counter electrode 53 and detecting electrode54 are formed on first insulating board 51. Those electrodes are made ofelectrically conductive material. Detecting electrode 54 in thisembodiment functions not only as an electrode for detecting ainsufficiency of a specimen but also as a part of a reference electrodeor as a part of the counter electrode.

FIG. 17 shows that the electrodes discussed above are disposed on thefirst insulating board; however, those electrodes may be divided anddisposed also on second insulating board 58 to be a cover board locatedopposite to first board 51.

Boards 51 and 58 are preferably made of polyethylene terephthalate,polycarbonate, polyimide or the like.

Each electrode is preferably made of electrically conductive materialsuch as noble metal including gold, platinum, and palladium, or simplematerial such as carbon. They may be also made of composite materialsuch as carbon paste or noble metal paste. In the former case, aconductive layer can be formed on board 51 or 58 easily by a sputteringevaporation method. In the latter case, a conductive layer can be formedon board 51 or 58 easily by a screen printing method.

The conductive layer is formed on an entire or a part of firstinsulating board 51 or second insulating board 58 by the sputteringevaporation method or the screen printing method. Then slits areprovided by laser for forming and dividing the electrodes. Theelectrodes may be formed by the screen printing method or a sputteringevaporation method on a printed board or a masked board having electrodepatterns formed in advance.

On the electrodes thus formed, reagent layer 55 is formed. Reagent layer35 includes enzymes, electron carriers and hydrophilic high-polymer. Theenzymes include glucose oxidase, lactate oxidase, cholesterol oxidase,cholesterol estrase, uricase, ascorbate acid oxidase, billrubin oxidase,glucose dehydrogenase, lactate dehydrogenase. The electron carrierpreferably employ ferricyanide kalium and may employ p-benzoquinone andits derivatives, phenacine methor sulphate, methylene blue, or pherocaneand its derivatives.

The hydrophilic high-polymer employ, e.g. carboxymethyl cellulose,hdroxy-ethyl cellulose, hydroxy propyl cellulose, methyl cellulose,ethyl cellulose, ethyl hydroxyethyl cellulose, carboxy methyl ethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polyamino acidssuch as poly-lysine, sulfonated polystyrene acid, gelatin and itsderivatives, acrylic acid and its salts, methacrylic acid and its salts,starch and its derivatives, anhydrous maleci acid and its salts, oragarose gel and its derivatives.

First insulating board 51 and second insulating board 58 are bonded viaspacer 56 in between for forming sample supplying path 57, from whichsample liquid is supplied. Spacer 56 has slit-shaped notch 57 formedtherein.

A significant difference from the conventional biosensor is that firstboard 51 and second board 58 forming path 57 are placed with their endsat an inlet of sample supplying path 57 deviated each other and bonded.That is, respective ends are placed at different places from each other.This preparation is viewed from a plan view. In other words, first board51 and second board 58 are in the same shape near the inlet of path 57;however, second board 58 and spacer 56 protrude toward the inlet withrespect to first board 51.

This allows the sample liquid to be sucked exactly and easily eventhough the sample supplying angle is small. This prevents the sampleliquid from attaching to the rear side of first board 51. Even if thesample liquid attaches to the rear side, the sample liquid can besupplied again smoothly.

The deviation of second board 58 from first board 51 at the endsthereof, that is, distance S1 between points 63 a and 64 a is preferablynot less than 0.1 mm and more preferably ranges from 0.25 to 1.0 mm,where center line L of path 57 shown in FIG. 18 crosses with first board51 and second board 58 at points 64 a and 63 a, respectively.

If being less than 0.1 mm, distance S1 is too short. The sample liquidthus cannot be supplied well if the sample supplying angle is small asin the conventional biosensor.

If first board 51 has different shape from second board 58 near theinlet of path 57 as shown in FIG. 19, a similar advantage to thatdiscussed above is measurable. In this case, the deviation at the endsthereof, i.e., center line L of path 57 shown in FIG. 20 crosses withfirst board 51 at point 64 b and crosses with second board 58 at point63 b. Distance S2 between point 63 b and point 64 b is preferably notless than 0.1 mm and more preferably ranging from 0.25 to 1.0 mm.

In the structures illustrated in FIG. 17 to FIG. 20, a depth of thesample supplying path, i.e., a thickness of spacer 56, ranges preferablyfrom 0.05 to 0.3 mm in order to supply the sample liquid quickly toslit-shaped path 57.

Spacer 56 is preferably made of polyethylene terephthalate,polycarbonate, polyimide, polybutylene terephthalate, polyamide,polyvinyl chloride, polyvinylidene chloride, or nylon.

For form sample supplying path 57, first board 51 may be bonded tosecond board 58 integrated with spacer 56 into one unit.

Reagent layer 55 is disposed on entire or a part of a surface of theelectrode, and however, may be disposed anywhere in sample supplyingpath 57 as long as it does not lower the performance of the biosensor.The sample liquid is supplied to the biosensor through path 57 having astructure discussed above by the capillary phenomenon. However, air hole59 through which air flows outside the biosensor is necessary in path 57in order to supply the sample liquid smoothly. Air hole 59 may shape ina rectangle, circle or polygon.

Air hole 59 may be located anywhere in path 57 as long as it does notblock the supply of sample liquid.

Hydrophilic treatment which may be performed inside path 57 enables thesample liquid to be supplied into path 57 more quickly and accurately.The hydrophilic treatment is realized by developing surface active agentinto or on second board 58, or by roughing the surface of the board bysand-blasting, electric-discharge machining, non-glare process, matprocess, or chemical plating.

In the biosensor discussed above, a current is generated by the reactionbetween a specific component in the sample liquid and reagent layer 55containing enzymes. The current is conducted to an external measuringinstrument (not shown) via lead-wires 60, 61, and 62 of measuringelectrode 52, counter electrode 53, detecting electrode 54 for beingmeasured.

For the current measurement, a triple-electrode method employingmeasuring electrode 52, counter electrode 53 and detecting electrode 54is available as discussed in this embodiment. Besides thetriple-electrode method, a double-electrode method employing onlymeasuring electrode 52 and counter electrode 53 is available. Eithermethod can produce the similar advantage to that of this embodiment;however, the triple-electrode method achieves more precise measurement.

EXAMPLE 1

A thin palladium film of 8 nm thickness was formed on the entire surfaceof the first insulating board made of polyethylene terephthalate bysputtering evaporation method. Then slits were provided on a part of thethin film by YAG laser, and thus the electrode was divided into ameasuring electrode, a counter electrode and a detecting electrode. Ontop of that, water solution containing enzymes, electron carriers, andhydrophilic high-polymer was dripped such that the water solutioncovered the measuring electrode as a center and parts of the counterelectrode as well as the detecting electrode. Then the water solutionwas dried to form a reagent layer. Further on top of that, a spacer madeof polyethylene terephthalate and having a notch together with thesecond insulating board (cover) made of polyethylene terephthalate andhaving the air hole was bonded. As a result, the sample supplying path,i.e., a capillary which leads blood, was formed.

In order to confirm the advantage of the present invention, thefollowing six types of blood-sugar value sensors having end-deviations(distance S) from the board to the spacer and cover were determined as:S=0 (a conventional sensor), 0.1, 0.25, 0.5, 1.0, and 2.0 mm.

Surface active agent is applied to the surface of the cover (inside ofthe sample supplying path) in order to supply the blood to the path morequickly. FIG. 21 illustrates a test method for confirming ablood-sucking performance of the sensor depending on a blood-supplyingangle in the blood-sugar value sensor discussed above. Table 1 shows thetest result.

TABLE 1 S Blood Supply (mm) Angle (deg.) 1 2 3 4 5 Conventional 0 0 x xx x x Sensor 15 Δ x x Δ x 30 Δ Δ Δ x Δ 45 ∘ ∘ ∘ ∘ Δ 90 ∘ ∘ ∘ ∘ ∘ SensorAccording 0.1 0 ∘ Δ Δ ∘ Δ to Present 15 ∘ Δ ∘ ∘ Δ Invention 30 ∘ ∘ ∘ ∘ Δ45 ∘ ∘ ∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 0.25 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45∘ ∘ ∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 0.5 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 1.0 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘ ∘ ∘∘ 90 ∘ ∘ ∘ ∘ ∘ 2.0 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘ ∘ ∘ ∘ 90∘ ∘ ∘ ∘ ∘ Definitions of the marks in the table: ∘: The blood is suckedby one sucking. Δ: The blood is sucked by two or three suckingoperations. x: The blood is not sucked at all.

Table 1 tells that the conventional sensor having distance S=0 mm doesnot suck blood and requires several trials of supply for proper suckingwhen it has a small blood-supplying angle (0-30 degree). For a smallblood-supplying angle, when a user supplies the blood to the samplesupplying path, the blood attaches to the rear side of the insulatingboard firstly. Thus even if the user tries to supply the blood again,the blood is pulled by the blood attached to the rear side. This may bea reason why the conventional sensor does not work well.

The sensor of the present invention, on the other hand, sometimesrequires several sucking operations when the blood-supplying angle issmall even at the shortest distance S=0.1 mm; however the sensor doessuck the blood at all. When the distance S is not less than 0.25 mm, thesensor sucks the blood easily at any sucking angle.

FIG. 22 illustrates a test method for testing the sensor in theblood-sucking performance depending on the blood-supplying angle. Inthis test, blood is attached to the rear side of the insulating board inadvance at an area of 5 mm from the end of the board in order to preventthe blood from being sucked. Table 2 shows the test result.

TABLE 2 S Blood Supply (mm) Angle (deg.) 1 2 3 4 5 Conventional 0 0 x xx x x Sensor 15 x x x x x 30 x x x x x 45 x x x x x 90 x x x x x SensorAccording 0.1 0 x Δ Δ Δ x to Present 15 Δ Δ Δ x Δ Invention 30 Δ ∘ Δ ∘ Δ45 ∘ ∘ ∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 0.25 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45∘ ∘ ∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 0.5 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘∘ ∘ ∘ 90 ∘ ∘ ∘ ∘ ∘ 1.0 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘ ∘ ∘∘ 90 ∘ ∘ ∘ ∘ ∘ 2.0 0 ∘ ∘ ∘ ∘ ∘ 15 ∘ ∘ ∘ ∘ ∘ 30 ∘ ∘ ∘ ∘ ∘ 45 ∘ ∘ ∘ ∘ ∘ 90∘ ∘ ∘ ∘ ∘ Definitions of the marks in the table: ∘: The blood is suckedby one sucking. Δ: The blood is sucked by two or three suckingoperations. x: The blood is not sucked at all.

Table 2 shows that the conventional sensor having the distance S=0 mmcan not suck the blood except the blood-supplying angle of 90 degree. Onthe other hand, the sensor of the present invention sometimes cannotsuck the blood when distance S=0.1 mm at a small blood-supplying angle.However, the sensor can suck the blood easily at any blood-supplyingangle when distance S is not less than 0.25 mm.

According to the second embodiment discussed above, the respective endsof first insulating board 51 and second insulating board 58 are deviatedeach other so that both the ends are placed at different places viewedfrom a plan view. This allows the sample liquid to be sucked exactly andeasily.

In the second embodiment, an enzyme sensor as the biosensor isdescribed. The present invention is similarly applicable to biosensorsincluding a molecular recognition element reacting not only with theenzyme but also with germ, antibody, DNA, or RNA.

According to the sensor in accordance with the second embodiment, twoboards bonded together form the sample supplying path, from which thesample liquid is taken out, between the boards. An opening is providedas an inlet at respective ends of both boards for accepting the sampleliquid. The ends forming the inlet are located at different places orshaped in different forms viewed from a plan view of the biosensor. Thusthe supply sample liquid can be sucked exactly and easily even if thesample-supplying angle is not enough (small). Further this prevents thesample liquid from attaching to the rear side of first insulating board51. If the sample liquid attaches to the rear side, a user can supplythe sample liquid again to allowing the sample liquid to be suppliedsmoothly.

INDUSTRIAL APPLICABILITY

The present invention provides a biosensor which is handled by a usereasily and exhibits an excellent measurement accuracy. The presentinvention also provides a measuring method using the biosensor as wellas a measuring device using the biosensor.

1. A method of measuring a quantity of a substrate included in a sampleliquid, comprising the steps of: providing a biosensor which includes:an insulating board; an electrode section formed on at least a part ofthe insulating board, the electrode section including a counterelectrode, a measuring electrode, and a detecting electrode: a samplesupplying path for supplying the sample liquid to the electrode section;and a reagent layer for reacting with the sample liquid supplied throughthe sample supplying path, wherein the detecting electrode is disposedmost downstream among the counter electrode, the measuring electrode,and the detecting electrode along the sample supplying path from aninlet along a direction of flow of the sample liquid; providing ameasuring device which includes: a supporting section for detachablysupporting the biosensor; and a connecting terminal and a driving powersupply for applying a voltage to the electrode section; inserting thebiosensor into the supporting section of the measuring device; applyinga voltage with the driving power supply to a first group including thecounter electrode and the measuring electrode; and applying a voltagewith the driving power supply to a second group including the detectingelectrode and one of the measuring electrode and the counter electrode;and determining whether or not the sample liquid is suppliedsufficiently for measurement depending on whether or not respectiveelectric currents supplied from the first and second groups exceedrespective predetermined thresholds.
 2. The method of claim 1, whereinsaid step of determining whether or not the sample liquid is suppliedsufficiently comprises the sub-step of: determining that the sampleliquid is insufficient when the electric current from the second groupdoes not exceed the predetermined threshold within a given lapse of timesince the electric current from the first group exceeds thepredetermined threshold.
 3. The method of claim 2, further comprisingthe step of: when it is determined that the sample liquid isinsufficient, outputting the determination to outside of the measuringdevice.
 4. The method of claim 1, further comprising the step of:halting steps of measuring in order to add the sample liquid when theelectric current from the second group does not exceed the predeterminedthreshold within a given lapse of time since the electric current fromthe first group exceeds the predetermined threshold.
 5. The method ofclaim 1, wherein an air hole is provided more downstream than thedetecting electrode for accelerating the flow of the sample liquid, saidmethod further comprising the step of: determining that the sampleliquid is sucked from the air hole by mistake when the electric currentfrom the second group exceeds the predetermined threshold before theelectric current from the first group exceeds the predeterminedthreshold and when the electric current from the first group does notexceed the predetermined threshold within a given lapse of time.
 6. Themethod of claim 1, further comprising the step of: compensating ameasured quantity of the substrate corresponding to an electric currentdetected at the electrode section depending on a lapse of time since theelectric current from the first group exceeds the predeterminedthreshold until the electric current from the second group exceeds thepredetermined threshold.
 7. The method of claim 6, wherein the measuringdevice further includes a memory for storing calibration data indicatingcorrespondence between an electric current detected at the biosensor anda quantity of the substrate in the sample liquid, said method furthercomprising the step of: determining a quantity of the substratecorresponding to the detected electric current referring to thecalibration data stored in the memory.
 8. The method of claim 1, furthercomprising the step of: incubating reaction between the sample liquidand the reagent layer after the sample liquid is supplied to the samplesupplying path; and varying a time for said step of incubating thereaction according to a lapse of time since the electric current fromthe first group exceeds the predetermined threshold until the electriccurrent from the second group exceeds the predetermined threshold. 9.The method of claim 1, further comprising: switching the voltage appliedto one of the first and second groups at a given interval.